U.S. patent application number 14/540722 was filed with the patent office on 2015-05-14 for methods of detecting cells latently infected with hiv.
This patent application is currently assigned to Oregon Health and Science University. The applicant listed for this patent is Oregon Health and Science University. Invention is credited to Nicolas Chomont, Remi FROMENTIN, Rafick-Pierre Sekaly.
Application Number | 20150132744 14/540722 |
Document ID | / |
Family ID | 53044097 |
Filed Date | 2015-05-14 |
United States Patent
Application |
20150132744 |
Kind Code |
A1 |
FROMENTIN; Remi ; et
al. |
May 14, 2015 |
METHODS OF DETECTING CELLS LATENTLY INFECTED WITH HIV
Abstract
Disclosed are methods of detecting and isolating CD4.sup.+ T
cells latently infected with HIV as well as methods of screening
for inhibitors of CD4.sup.+ T cells latently infected with HIV.
Inventors: |
FROMENTIN; Remi; (Port St.
Lucie, FL) ; Chomont; Nicolas; (Port St. Lucie,
FL) ; Sekaly; Rafick-Pierre; (Cleveland, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Oregon Health and Science University |
Portland |
OR |
US |
|
|
Assignee: |
Oregon Health and Science
University
Portland
OR
|
Family ID: |
53044097 |
Appl. No.: |
14/540722 |
Filed: |
November 13, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61903799 |
Nov 13, 2013 |
|
|
|
Current U.S.
Class: |
435/5 |
Current CPC
Class: |
G01N 2500/10 20130101;
G01N 33/505 20130101; G01N 2333/70596 20130101; G01N 33/56972
20130101; G01N 2333/70514 20130101; G01N 33/56988 20130101 |
Class at
Publication: |
435/5 |
International
Class: |
G01N 33/569 20060101
G01N033/569; G01N 33/50 20060101 G01N033/50 |
Goverment Interests
ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with United States government
support under the terms of grant number U19 AI096109, awarded by
the National Institutes of Health. The United States government has
certain rights in this invention.
Claims
1. A method of identifying a latently infected CD4.sup.+ T cell
from a subject, the method comprising: obtaining a biological
sample from the subject, the biological sample comprising CD4.sup.+
T cells; contacting the sample with a first labeled antibody
comprising a first label, wherein the first labeled antibody
specifically binds PD-1, LAG-3, or TIGIT; forming a complex between
the first labeled antibody to PD-1, LAG-3, or TIGIT with PD-1,
LAG-3, or TIGIT; detecting the complex by detecting the first label
on the surface of a CD4.sup.+ cell that has bound the labeled
antibody in the complex; wherein detection of the first label
indicates that the cell is a latently infected CD4.sup.+ T
cell.
2. The method of claim 1 wherein detecting the first label is
performed using flow cytometry.
3. The method of claim 1 further comprising contacting the sample
with a second labeled antibody, wherein the second labeled antibody
binds CD4.
4. The method of claim 1 further comprising isolating the CD4.sup.+
T cells that have bound the first antibody.
5. The method of claim 4 wherein isolating further comprises
fluorescently activated cell sorting or magnetic sorting.
6. A population of CD4.sup.+ T cells isolated by the method of
claim 4 or 5.
7. The population of CD4.sup.+ T cells from claim 6 wherein the
population is isolated from a subject undergoing antiretroviral
therapy.
8. A method of selecting a compound that inhibits a latently
infected CD4.sup.+ T cell, the method comprising: contacting the
CD4.sup.+ T cell of claim 6 or 7 with a test compound; assessing
whether or not the test compound inhibited a CD4.sup.+ T cell of
claims 6-7.
9. The method of claim 8 further comprising contacting a control
CD4.sup.+ T cell with the test compound wherein the control
CD4.sup.+ T cell is known not to comprise latent HIV nucleic
acid.
10. The method of claim 9 wherein the control CD4.sup.+ T cell is a
CD4.sup.+ T cell from an HIV-negative subject.
11. The method of claim 9 wherein the test compound comprises an
antibody specific for PD-1, LAG-3, or TIGIT.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 61/903,799, filed Nov. 13, 2013, the entirety of
which is incorporated by reference herein.
BACKGROUND
[0003] Antiretroviral therapy (ART) reduces HIV replication but
does not cure HIV. The persistence of HIV in a small pool of
long-lived latently infected resting CD4 T cells is a major barrier
to viral eradication. Methods of identifying cells in which HIV
persists are clearly necessary.
SUMMARY
[0004] Several immune checkpoint blockers (ICBs) have been shown to
actively reduce T cell activation, proliferation and cytokine
production in CD4.sup.+ T cells, thereby acting as negative
regulators of T-cell activation. In particular, PD-1, LAG-3 and
TIGIT have been identified as markers associated with incomplete
CD4 T cell restoration and HIV persistence during ART. Disclosed
herein are methods involving the surprising discovery relating to
identifying a cell comprising a latent HIV nucleic acid by
detecting one or more of PD-1, LAG-3 and TIGIT, alone or in
combination with each other or any other marker.
[0005] It is an object of the invention to efficiently identify
cells latently infected with HIV.
[0006] It is an object of the invention to allow efficient sorting
of cells latently infected with HIV.
[0007] It is an object of the invention to provide a population of
cells that can be used to screen test compounds for potential
therapeutics that inhibit (or eliminate) cells latently infected
with HIV.
[0008] The methods involve obtaining a biological sample from a
subject, the biological sample comprising CD4.sup.+ T cells. The
sample is contacted with a labeled antibody that is conjugated to
the label. The labeled antibody is specific for PD-1, AG-3, or
TIGIT. The label can be detected on the surface of a CD4.sup.+ T
cell that has bound the labeled antibody, thereby indicating that
the CD4.sup.+ T cell expresses PD-1, LAG-3, and/or TIGIT on its
surface. Detection of the label indicates that the cell comprises
latent HIV nucleic acid.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 is a plot of the expression of the indicated immune
checkpoint blockers PD-1, LAG-3, TIGIT, CTLA-4, BTLA, CD160, 2B4,
and TIM-3 on CD4.sup.+ T cells isolated from HIV infected subjects
undergoing antiretroviral therapy.
[0010] FIG. 2 shows the association between CD4 count and the
frequency of CD4 T cells expressing PD-1, LAG-3, and TIGIT. Panel A
shows a plot of total CD4 T cell count against the percentage of
PD-1.sup.+ T cells isolated from HIV infected subjects undergoing
antiretroviral therapy. Panel B shows a plot of total CD4 T cell
count against the percentage of LAG-3.sup.+ T cells isolated from
HIV infected subjects undergoing antiretroviral therapy. Panel C
shows a plot of total CD4 T cell count against the percentage of
TIGIT.sup.+ T cells isolated from HIV infected subjects undergoing
antiretroviral therapy.
[0011] FIG. 3 is a plot showing copy number of integrated HIV DNA,
total HIV DNA, 2-LTR HIV DNA and unspliced HIV RNA from HIV
infected subjects undergoing antiretroviral therapy.
[0012] FIG. 4 shows that the frequency of CD4 T cells harboring
intergrated HIV DNA was positively correlated with the expression
of PD-1, LAG-3, and TIGIT. Panel A shows a plot of PD-1.sup.+;
CD4.sup.+ T cells against integrated HIV DNA copy number per
10.sup.6 CD4 T cells from HIV infected subjects undergoing
antiretroviral therapy. Panel B shows a plot of LAG-3.sup.+;
CD4.sup.+ T cells against integrated HIV DNA copy number per
10.sup.6 CD4 T cells from HIV infected subjects undergoing
antiretroviral therapy, Panel C shows a plot of TIGIT.sup.+
CD4.sup.+ T cells against integrated HIV DNA copy number per
10.sup.6 CD4 T cells from HIV infected subjects undergoing
antiretroviral therapy.
DETAILED DESCRIPTION
Definitions
[0013] For the purpose of the current disclosure, the following
definitions shall, in their entireties, be used to define technical
terms, and to define the scope of the composition of matter for
which protection is sought in the claims.
[0014] Antibody:
[0015] A polypeptide including at least a light chain or heavy
chain immunoglobulin variable region which specifically recognizes
and binds an epitope of an antigen or a fragment thereof,
Antibodies are composed of a heavy and a light chain, each of which
has a variable region, termed the variable heavy (VH) region and
the variable light (VL) region. Together, the VH region and the VL
region are responsible for binding the antigen recognized by the
antibody.
[0016] The term "antibody" encompasses intact immunoglobulins, as
well the variants and portions thereof, such as Fab fragments, Fab'
fragments, F(ab)'2 fragments, single chain Fv proteins ("scFv"),
and disulfide stabilized RI proteins ("dsFv"). In some aspects, the
antibody can be a camelid-derived antibody. In some aspects, the
antibody can be an antibody derived from cartilaginous fishes. A
scFv protein is a fusion protein in which a light chain variable
region of an immunoglobulin and a heavy chain variable region of an
immunoglobulin are bound by a linker. In dsFvs the chains have been
mutated to introduce a disulfide bond to stabilize the association
of the chains. The term also includes genetically engineered forms
such as chimeric antibodies, and heteroconjugate antibodies (such
as, bispecific antibodies). See also, Pierce Catalog and Handbook,
Pierce Chemical Co., Rockford, Ill., 1994-1995; Kuby, J.,
Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997. The
term includes both monoclonal and polyclonal types of antibodies,
One or more monoclonal antibody species may be combined. An
antibody of the present invention may be recombinant or produced
using hybridoma technology.
[0017] The PD-1, LAG-3 and TIGIT binding antibodies can be
full-length. In some aspects, the full-length antibodies can be
selected from one of the following antibody types: IgG (as
non-limiting examples: an IgG1, IgG2, IgG3, IgG4), IgM, and IgA (as
non-limiting examples: IgA1, or IgA2), IgO, and IgE). In some
aspects, the PD-1, LAG-3 and TIGIT binding antibodies can comprise
an antigen-binding fragment (as non-limiting examples: a Fab, Fab',
F(ab'h or scFv fragment). In some embodiments, the antigen-binding
fragment does not need to include an Fe domain or a CH2, CH3, or
CH4 sequence. The antibody can include two heavy chain
immunoglobulins and two light chain immunoglobulins, or can be a
single chain antibody. The antibodies can, optionally, include a
constant region chosen from a kappa, lambda, alpha, gamma, delta
epsilon or a mu constant region gene. A PD-1, LAG-3 and TIGIT
marker of the present invention-binding antibody can include a
heavy and light chain constant region substantially from a human
antibody, as a non-limiting example a human IgG1 constant region or
a portion thereof, or from another species, including but not
limited to, mouse, rat, dog, cat, goat, sheep, cow, horse, chicken
or guinea pig.
[0018] In one embodiment, the antibody (or fragment thereof) is a
recombinant or modified antibody. In some aspects, the recombinant
or modified antibody can be a chimeric, a humanized, a deimmunized,
or an in vitro generated antibody. The term "recombinant" or
"modified" antibody, as used herein, is intended to include all
antibodies that are prepared, expressed, created or isolated by
recombinant means, such as antibodies expressed using a recombinant
expression vector transfected into a host cell, antibodies isolated
from a recombinant, combinatorial antibody library, antibodies
isolated from an animal that is transgenic for human immunoglobulin
genes or antibodies prepared, expressed, created or isolated by any
other means that involves spacing of immunoglobulin gene sequences
to other DNA sequences. Such recombinant antibodies include
humanized, CDR grafted, chimeric, deimmunized, in vitro generated
antibodies, and may optionally include constant regions derived
from human germane immunoglobulin sequences.
[0019] The VH and VL regions can be further subdivided into regions
of hypervariability, termed "complementarity determining regions"
(CDR), interspersed with regions that are more conserved, termed
"framework regions" (FR). The extent of the framework region and
CDRs has been precisely defined (see, Kabat, E. A, et al.,
Sequences of Proteins of immunological interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publication No.
91-3242, 1991, and Chothia, C. et al., J. Mol. Biol., 196:901-917,
1987). Kabat definitions are used herein. Each VH and VL is
typically composed of three CDRs and four FRs, arranged from
amino-terminus to carboxy-terminus in the following order: FR1,
CDR1, FR2, CDR2, FR3, CDR3, FR4.
[0020] An "immunoglobulin domain" refers to a domain from the
variable or constant domain of immunoglobulin molecules.
Immunoglobulin domains typically contain two beta-sheets formed of
about seven beta-strands, and a conserved disulphide bond (see, A.
F. Williams and A. N. Barclay, Ann. Rev Immunol., 6:381-405, 1988).
The canonical structures of hypervariable loops of an
immunoglobulin variable can be inferred from its sequence, as
described in Chothia et al., J Mol. Biol., 227:799-5 817, 1992;
Tomlinson et al., J. Biol., 227:776-798, 1992; and Tomlinson et
al., EMBO J., 14(18):4628-38, 1995.
[0021] As used herein, an "immunoglobulin variable domain sequence"
refers to an amino acid sequence which can form the structure of an
immunoglobulin variable domain. As a non-limiting example, the
sequence may include all or part of the amino acid sequence of a
naturally-occurring variable domain. The sequence may omit one, two
or more N- or C-terminal amino acids, internal amino acids, may
include one or more insertions or additional terminal amino adds,
or may include other alterations. In one embodiment, a polypeptide
that includes immunoglobulin variable domain sequence can associate
with another immunoglobulin variable domain sequence to form a
target binding structure (or "antigen binding site"). In some
aspects, the antigen binding site may be a structure that interacts
with a PD-1, LAG-3 or TIGIT marker of the present invention. In
some aspects, the interaction can be binding or inhibiting.
[0022] The VH or VL chain of the antibody can further include a or
part of a heavy or light chain constant region, to thereby form a
heavy or light immunoglobulin chain, respectively. In one
embodiment, the antibody can be a tetramer of two heavy
immunoglobulin chains and two light immunoglobulin chains, wherein
the heavy and light immunoglobulin chains are inter-connected by
disulfide bonds. The heavy chain constant region includes three
domains, CH1, CH2 and CH3. The light chain constant region includes
a CL domain. The variable region of the heavy and light chains
contains a binding domain that interacts with an antigen. The
constant regions of the antibodies typically mediate the binding of
the antibody to a host tissue or factors, including various cells
of the immune system (as a non-limiting example, effector cells)
and the first component of the classical complement system. The
term "antibody" includes intact immunoglobulins of types IgA, IgG,
IgE, IgD, IgM (as well as subtypes thereof). In one embodiment the
antibody can be an IgA, In another embodiment the antibody can be
an IgG. In another embodiment the antibody can be an IgE. In
another embodiment the antibody is an IgD. In another embodiment
the antibody can be an IgM. The light chains of the immunoglobulin
may be of types kappa or lambda. In one embodiment, the antibody
can be glycosylated.
[0023] One or more regions of an antibody can be human or
effectively human. As a non-limiting example, one or more of the
variable regions can be human or effectively human. As a
non-limiting example, one or more of the CDRs can be human. In some
aspects, the human CDRs may be HC CDR1., HC CDR2HC CDR3, LC CDR1,
LC CDR2, or LC CDR3. Each of the light chain CDRs can be human. HC
CDR3 can be human. One or more of the framework regions can be
human. In some aspects, the human framework regions can be FR1,
FR2FR3, and FR4 of the HC or LC. In some aspects, all the framework
regions are human. In some aspects, the frameworks regions are
derived from a human somatic cell. In some aspects, the human
somatic cell is a hematopoietic cell that produces immunoglobulins
or a non-hematopoietic cell. In some embodiments, the human
sequences are germline sequences. In some embodiments, the germline
sequences are encoded by a germline nucleic acid.
[0024] One or more of the constant regions can be human or
effectively human. In some embodiments, at least 70, 75, 80, 85,
90, 92, 95, or 98% of the framework regions (FR1, FR2, and FR3,
collectively, or FR1, FR2, FR3, and FR4, collectively) or the
entire antibody can be human or effectively human. As a
non-limiting example, FR1, FR2, and FR3 collectively can be at
least 70, 75, 80, 85, 90, 92, 95, 98, or 99% identical to a human
sequence encoded by a human germline V segment of a locus encoding
a light or heavy chain sequence.
[0025] All or part of an antibody can be encoded by an
immunoglobulin gene or a segment thereof. Exemplary immunoglobulin
genes include the kappa, lambda, alpha (IgA1 and IgA2), gamma
(IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu constant region
genes, as well as the myriad immunoglobulin variable region genes.
Full-length immunoglobulin light chains can be encoded by a
variable region gene at the NH2-terminus and a kappa or lambda
constant region gene at the COOH-terminus. Full-immunoglobulin
heavy chains can be similarly encoded by a variable region gene and
one previously mentioned constant region genes. A light chain
refers to any polypeptide that includes a light chain variable
domain. A heavy chain refers to any polypeptide that a heavy chain
variable domain.
[0026] The term "antigen-binding fragment" of a full-length
antibody (or simply "antibody portion," or "fragment"), as used
herein, refers to one or more fragments of a full-length antibody
that retain the ability to specifically bind to a target of
interest. Examples of binding fragments encompassed within the term
"antigen-binding fragment" of a full length antibody include (i) a
Fab fragment, a monovalent fragment consisting of the VL, VH, CL
and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment
including two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fd fragment consisting of the VH and CH1
domains; (iv) a Fv fragment consisting of the VL and VH domains of
a single arm of an antibody, (v) a dAb fragment (Ward et al.,
Nature, 341:544-546, 1989), which consists of a VH domain; and (vi)
an isolated complementarity determining region (CDR) that retains
functionality. Furthermore, although the two domains of the Fv
fragment, VL and VH, are coded for by separate genes, they can be
joined, using recombinant methods, by a synthetic linker that
enables them to be made as a single protein chain in which the VL
and VH regions pair to form monovalent molecules known as single
chain Fv (scFv). See Bird et al., Science, 242:423-426, 1988; and
Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883, 1988.
[0027] A "humanized" immunoglobulin variable region can be an
immunoglobulin variable region that includes sufficient number of
human framework amino acid positions such that the immunoglobulin
variable region does not elicit an immunogenic response in a normal
human. Descriptions of "humanized" immunoglobulins include, for
example, U.S. Pat. No. 6,407,213 and U.S. Pat. No. 5,693,762.
[0028] An "effectively human" immunoglobulin variable region is an
immunoglobulin variable region that includes a sufficient number of
human framework amino acid positions such that the immunoglobulin
variable region does not elicit an immunogenic response in a normal
human. An "effectively human" antibody is an antibody that includes
a sufficient number of human amino acid positions such that the
antibody does not elicit an immunogenic response in a normal
human.
[0029] As used herein, "binding affinity" refers to the apparent
association constant or Ka, Binding affinity may be expressed as
the dissociation constant (Kd) which is the reciprocal of the Ka. A
target binding agent, such as an antibody may, for example, have a
Kd of less than 10.sup.-5, 10.sup.-6, 10.sup.-7 or 10.sup.-3 M for
a particular target molecule. Differences in binding affinity (for
specificity or other comparisons) can be at least 1.5, 2, 5, 10,
25, 50, 100, or 1 000-fold.
[0030] Binding affinity can be determined by a variety of methods
including equilibrium dialysis, equilibrium binding, gel
filtration, ELISA, surface plasmon resonance, or spectroscopy (as a
non-limiting example, a fluorescence assay). These techniques can
be used to measure the concentration of bound and free ligand as a
function of ligand (or target) concentration. The concentration of
bound ligand (Bound) is related to the concentration of free ligand
(Free and the concentration of binding sites for the ligand on the
target where (N) is the number of binding sites per target molecule
by the following equation:
(Bound)=N((Free)/((1/Ka)+(Free))
[0031] Although quantitative measurements of Ka are routine, it is
not always necessary to make an exact determination of Ka, though,
since sometimes it is sufficient to obtain a qualitative
measurement of affinity. Such qualitative measurements of affinity
can be determined using a method such as ELISA or FACS analysis, is
proportional to Ka, and thus can be used for comparisons, such as
determining whether a higher affinity is 2, 5, 10, 20, or 50 fold
higher than a reference.
[0032] Aptamers.
[0033] In some embodiments, the invention also features target
protein-binding agents such as aptamers. Aptamers may be nucleic
acid aptamers or peptide aptamers. The term "nucleic acid aptamer,"
as used herein, refers to a nucleic acid molecule which has a
conformation that includes an internal non-duplex nucleic acid
structure of at least 5 nucleotides. An aptamer can be a
single-stranded nucleic acid molecule which has regions of
self-complementarity. "Peptide aptamers" are short peptide
sequences presented and conformationally constrained in a robust,
inert protein scaffold (Evans et al., Journal of Biology, 7:3,
2008). The three-dimensional conformational constraint of the
inserted peptide applied by the protein scaffold readily increases
the affinity of the aptamer for the target over that of an
unconstrained peptide sequence. Exemplary aptamers include nucleic
acid molecules and peptides that bind to PD-1, LAG-3 or TIGIT
markers of the present invention. Particular aptamers may be used
in place of an antibody in many cases. Other peptides that bind a
PD-1, LAG-3 or TIGIT marker of the invention are also included.
Peptide-like molecules such as peptoids are further included in the
invention. "Peptoids", or poly-N-substituted glycines, are a class
of peptidomimetics whose side chains are appended to the nitrogen
atom of the peptide backbone, rather than to the alpha-carbons (as
they are in amino acids).
[0034] Binding Agent.
[0035] The term "binding agent" refers to an agent capable of
binding to a PD-1, LAG-3 or TIGIT marker of the present invention
under experimental conditions and include, but are not limited to,
antibodies and antigen-antibody binding fragments thereof,
including but not limited to Fab, Fab', F(ab'h, scFv or
single-domain antibody (sdAb), (also referred to as a nanobody),
nucleic acid aptamers, and peptide aptamers.
[0036] The PD-1, LAG-3 or TIGIT binding agents have in vitro and in
vivo diagnostic utilities. For example, measurement of levels of a
PD-1, LAG-3 or TIGIT marker in samples derived from a subject can
be used for the diagnosis of HIV. Moreover, the monitoring and
quantitation of a PD-1, LAG-3 or TIGIT marker level can be used
prognostically to stage the progression of HIV and to evaluate the
efficacy of agents used to treat a subject. Such a method can also
include contacting a reference sample (such as a "control sample")
with the binding agent, and determining the extent of formation of
the complex between the binding agent and the sample relative to
the same for the reference sample. A statistically significant
change in the formation of the complex in the sample or subject
relative to the control sample or subject can be indicative of the
presence of a latently infected CDC cell in the sample. The PD-1,
LAG-3, or TIGIT markers of the present invention-binding agent can
be directly or indirectly labeled with a detectable substance to
facilitate detection of the bound or unbound antibody. Suitable
detectable substances include various enzymes, prosthetic groups,
fluorescent materials, luminescent materials and radioactive
materials.
[0037] Antiretroviral Therapy:
[0038] A term that encompasses any of a number of treatment
regimens for subjects who have contracted HIV. Generally, these
treatment regimens include a combination of two or more
pharmaceutical compositions. Classes of pharmaceutical compositions
commonly used in antiretroviral therapy include viral entry
inhibitors, nucleoside/nucleotide reverse transcriptase inhibitors,
non-nucleoside reverse transcriptase inhibitors, integrase
inhibitors, and protease inhibitors.
[0039] Binding or Stable Binding:
[0040] An association between two substances or molecules, such as
the association of an antibody with a peptide, nucleic acid to
another nucleic acid, or the association of a protein with another
protein or nucleic acid molecule, or the association of a small
molecule drug with a protein (such as a tyrosine kinase) or other
biological macromolecule. Binding can be detected by any procedure
known to one skilled in the art, such as by physical or functional
properties. In some aspects, binding can be detected functionally
by determining whether binding has an observable effect upon a
biosynthetic process such as expression of a gene, DNA replication,
transcription, translation, protein activity (including tyrosine
kinase activity) and the like.
[0041] Biological Sample:
[0042] Obtaining a biological sample from a subject includes, but
need not be limited to any method of collecting a particular sample
known in the art. Obtaining a biological sample from a subject also
encompasses receiving a sample that was collected at a different
location than where a method is performed; receiving a sample that
was collected by a different individual than an individual that
performs the method, receiving a sample that was collected at any
time period prior to the performance of the method, receiving a
sample that was collected using a different instrument than the
instrument that performs the method, or any combination of these.
Obtaining a biological sample from a subject also encompasses
situations in which the collection of the sample and performance of
the method are performed at the same location, by the same
individual, at the same time, using the same instrument, or any
combination of these.
[0043] In some embodiments a biological sample can be selected
from: a solid tissue sample, blood, plasma, serum, sputum, or
urine. In some embodiments, the sample can be a blood sample.
[0044] A biological sample encompasses any fraction of a biological
sample or any component of a biological sample that may be isolated
and/or purified from the biological sample. For example: when cells
are isolated from blood or tissue, including specific cell types
sorted on the basis of biomarker expression; or when nucleic acid
or protein is purified from a fluid or tissue; or when blood is
separated into fractions such as plasma, serum, buffy coat PBMC's
or other cellular and non-cellular fractions on the basis of
centrifugation and/or filtration. A biological sample further
encompasses biological samples or fractions or components thereof
that have undergone a transformation of mater or any other
manipulation.
[0045] Biomarker:
[0046] Molecular, biological or physical attributes that
characterize a physiological or cellular state and that can be
objectively measured to detect or define disease progression or
predict or quantify therapeutic responses. A biomarker is a
characteristic that is objectively measured and evaluated as an
indicator of normal biologic processes, pathogenic processes, or
pharmacologic responses to a therapeutic intervention. A biomarker
may be any molecular structure produced by a cell or organism. A
biomarker may be expressed inside any cell or tissue; accessible on
the surface of a tissue or cell; structurally inherent to a cell or
tissue such as a structural component, secreted by a cell or
tissue, produced by the breakdown of a cell or tissue through
processes such as necrosis, apoptosis or the like; or any
combination of these. A biomarker may be any protein, carbohydrate,
fat, nucleic acid, catalytic site, or any combination of these such
as an enzyme, glycoprotein, cell membrane, virus, cell, organ,
organelle, or any uni- or multimolecular structure or any other
such structure now known or yet to be disclosed whether alone or in
combination.
[0047] A biomarker may be represented by the sequence of a nucleic
acid from which it can be derived or any other chemical structure.
Examples of such nucleic acids include miRNA, tRNA, siRNA, mRNA,
cDNA, or genomic DNA sequences including any complimentary
sequences thereof. One example of a biomarker is a DNA coding
sequence for a protein comprising one or more mutations that cause
amino acid substitutions in the protein sequence.
[0048] Contacting:
[0049] Placement in direct physical association, including
contacting of a solid with a solid, a liquid with a liquid, a
liquid with a solid, or either a liquid or a solid with a cell or
tissue, whether in vitro or in vivo. Contacting can occur in vitro
with isolated cells or tissue or in vivo by administering to a
subject.
[0050] FACS. (Fluorescent Activated Cell Sorting).
[0051] The PD-1., LAG-3, or TIGIT binding agent can be used to
label cells or protein. In some aspects, the cells or protein in a
biological sample can be a patient sample. The binding protein can
also be attached (or attachable) to a fluorescent compound. The
cells can then be sorted using fluorescent activated cell sorted.
As a non-limiting example, a fluorescent activated cell sorter can
be of the type available from Becton Dickinson Immunocytometry
Systems, San Jose Calif.; see also U.S. Pat. Nos. 5,627,037;
5,030,002; and 5,137,809), As cells pass through the sorter, a
laser beam excites the fluorescent compound while a detector counts
cells that pass through and determines whether a fluorescent
compound is attached to the cell by detecting fluorescence. The
amount of label bound to each cell can be quantified and analyzed
to characterize the sample. The sorter can also deflect the cell
and separate cells bound by the binding protein from those cells
not bound. The separated cells can be cultured and/or
characterized.
[0052] Inhibit:
[0053] To reduce to a measurable extent, for example, to reduce
activity of a CD4.sup.+ T cell latently infected with HIV.
Particular outcomes of inhibition include preventing latency from
reverting or promoting cell death (including programmed cell death)
of the CD4.sup.+ T cell latently infected with HIV. Preferably,
promoting cell death of the CD4.sup.+ T cell latently infected with
HIV does not involve promoting cell death of CD4.sup.+ T cells
lacking latent HIV nucleic acid.
[0054] Label:
[0055] A detectable compound or composition that can be conjugated
directly or indirectly to another molecule to facilitate detection
of that molecule. Specific, non-limiting examples of labels include
fluorescent tags, enzymatic linkages, and radioactive isotopes. In
some embodiments, the label can be a radioactive, fluorescent,
colorimeter or enzyme label. In some embodiments, the label can be
a second antibody that immunospecifically binds to the anti-PD-1,
anti-LAG-3 or anti-TIGIT antibodies. In some examples, a label can
be attached to an antibody to facilitate detection of the molecule
that the antibody specifically binds.
[0056] The terms "labeled antibody" or "tagged antibody", as used
herein, includes antibodies that are labeled by detectable means
and include, but are not limited to, antibodies that are
fluorescently, electroluminescently enzymatically, radioactively,
optical (non-limiting examples are plasmonic resonance,
lifetime-based, light scattering), magnetic, or chemiluminescently
labeled. Antibodies can also be labeled with a detectable tag, such
as c-Myc, HA, VSV-G, HSV, FLAG, V5, or HIS, which can be detected
using an antibody specific to the tag, for example, an anti-c-Myc
antibody. Antibodies can also be labeled with an enzyme. In some
embodiments, the enzyme can be alkaline phosphatase, acid
phosphatase, horseradish peroxidase, betagalactosidase or
ribonuclease. Antibodies can also be labelled with quantum dots
(nanoparticles) which exhibit quantum confinement effects and are
therefore subject to stimulated emission such as fluorescence or
electroluminescence. Various methods of labeling binding agents are
known in the art and may be used. Non-limiting examples of
fluorescent labels or tags for labeling the antibodies for use in
the methods of invention include QDot605, Brilliant Violet 650,
Violet 500, AmCyan, PerCP-eFluor 710, Hydroxycoumarin, Succinimidyl
ester, Aminocoumarin, Succinirnidyl ester, Methoxycoumarin,
Succinimidyl ester, Cascade Blue, Hydrazide Pacific Blue,
Maleimide, Pacific Orange, Lucifer yellow, NBD, NBD-X,
R-Phycoerythrin (PE), a PE-Cy5 conjugate (Cychrome, R670,
Tri-Color, Quantum Red), a PE-Cy7 conjugate, Red 613, PE-Texas Red,
PerCP, Peridinin chlorphyll protein, TruRed (PerCP-Cy5.5
conjugate), FluorX, Fluoresceinisothyocyanate, (FITC), BODIPY-FL,
TRITC, X-Rhodamine (XRJTC), Lisamine Rhodamine B, Texas Red,
Allophycocyanin (APC), APC-Cy7 conjugate, Alexa Fluor 350, Alexa
Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa
Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa
Fluor 568, Alexa Fluor 594, Alexa Fluor 610, 20 Alexa Fluor 633,
Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 674, Alexa Fluor 680,
Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, Cy2, Cy3, Cy38,
Cy3.5, Cy5, Cy5.5 or Cy7. A variety of suitable fluorescent species
and chromophores are described by Stryer, Science, 162:526, 1968
and Brand, L. et al., Annual Review of Biochemistry, 41:843-868,
1972. The binding proteins can be labeled with fluorescent or
chromophore groups by conventional procedures such as those
disclosed in U.S. Pat. Nos. 3,940,475, 4,289,747, and 4,376,110. In
some embodiments the fluorescent species can be a xanthene dye,
which include the fluoresceins and rhodamines. In some embodiments
the fluorescent compounds can be a naphthylamine, Once labeled with
a fluorophore or chromophore, the binding protein can be used to
detect the presence or localization of the PD-1, LAG-3, or TIGIT
markers of the present invention in a sample using fluorescent
microscopy. In some embodiments the fluorescent microscopy is
confocal or deconvolution microscopy, A bioluminescent compound may
be used to label the PD-1, LAG-3, or TIGIT markers. The presence of
a bioluminescence protein can be determined by detecting the
presence of luminescence. Important bioluminescence compounds for
purposes of labeling are luciferin, luciferase and aquorin. Other
methods of detecting include, but are not limited to, Biacore
(surface plasmon resonance), ELISA, histology, and
cell-staining.
[0057] In some embodiments of the aspects described herein, an
agent specific for a PD-1, LAG-3, or TIGIT marker, such as an
antibody or antigen-binding fragment thereof, a natural or
recombinant ligand, a small molecule, or a modifying moiety, is
directly labeled with a tag to facilitate the detection of the
modification. The terms "label" or "tag", as used herein, refer to
a composition capable of producing a detectable signal indicative
of the presence of a target, such as, the presence of a specific
modification in a biological sample. Suitable labels include
fluorescent molecules, radioisotopes, nucleotide chromophores,
enzymes, substrates, chemiluminescent moieties, magnetic particles,
bioluminescent moieties, peptide tags (c-Myc, HA, VSV-G, HSV, FLAG,
V5 or HIS) and the like. As such, a label is any composition
detectable by spectroscopic, photochemical, biochemical,
immunochemical, electrical, optical or chemical means needed for
the methods to identify the PD-1, LAG-3, or TIGIT markers. In some
embodiments of the aspects described herein, the modification
moiety itself may be labeled directly. As a non-limiting example,
one can use a radioactive label or a fluorescent label so that the
protein modification can be read directly (or in combination with
other modifications) without the use of antibodies.
[0058] Latent HIV Nucleic Acid:
[0059] Any form of HIV that is present within the CD4.sup.+ T cell
pool of an HIV infected subject, particularly an HIV infected
subject who is currently undergoing antiretroviral therapy. Latent
HIV nucleic acid can take many forms including HIV DNA that has
integrated in the genome and unspliced HIV RNA.
[0060] Polypeptide:
[0061] Any chain of amino acids, regardless of length or
posttranslational modification (such as glycosylation, methylation,
ubiquitination, phosphorylation, or the like). "Polypeptide" is
used interchangeably with "protein," and is used to refer to a
polymer of amino acid residues. A "residue" refers to an amino acid
or amino acid mimetic incorporated in a polypeptide by an amide
bond or amide bond mimetic.
[0062] Sample:
[0063] A sample, such as a biological sample, is a sample obtained
from a human or animal subject, such as a sample comprising
CD4.sup.+ T cells. Samples include, but are not limited to, cells,
tissues, and bodily fluids, including tissues that are, for
example, unfixed, frozen, fixed in formalin and/or embedded in
paraffin.
[0064] Sequence Identity/Similarity:
[0065] The identity/similarity between two or more nucleic acid
sequences, or two or more amino acid sequences, is expressed in
terms of the identity or similarity between the sequences. Sequence
identity can be measured in terms of percentage identity; the
higher the percentage, the more identical the sequences are.
Sequence similarity can be measured in terms of percentage
similarity (which takes into account conservative amino acid
substitutions); the higher the percentage, the more similar the
sequences are.
[0066] Methods of alignment of sequences for comparison are well
known in the art. Various programs and alignment algorithms are
described in: Smith & Waterman, Adv. Appl, Math., 2:482, 1981;
Needleman & Wunsch, J. Mol. Biol., 48:443, 1970; Pearson &
Lipman, Proc, Natl. Acad. Sci. USA, 85:2444, 1988; Higgins &
Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS, 5:151-3,
1989; Corpet et al., Nuc. Acids Res., 16:10881-90, 1988; Huang et
al., Computer Appls. in the Biosciences, 8, 155-65, 1992; and
Pearson et al., Meth. Mol. Bio., 24:307-31, 1994. Altschul et al.,
J. Mol. Biol., 215:403-10, 1990, presents a detailed consideration
of sequence alignment methods and homology calculations.
[0067] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul
et al., J. Mol, Biol., 215:403-10, 1990) is available from several
sources, including the National Center for Biological Information
(NCBI, National Library of Medicine, Building 38A, Room 8N805,
Bethesda, Md. 20894) and on the Internet, for use in connection
with sequence analysis programs blastp, blastn, blastx, tblastn and
tblastx. Additional information can be found at the NCBI web site.
BLASTN is used to compare nucleic acid sequences, while BLASTP is
used to compare amino acid sequences. If the two compared sequences
share homology, then the designated output file will present those
regions of homology as aligned sequences. If the two compared
sequences do not share homology, then the designated output file
will not present aligned sequences.
[0068] Once aligned, the number of matches is determined by
counting the number of positions where an identical nucleotide or
amino acid residue is presented in both sequences. The percent
sequence identity is determined by dividing the number of matches
either by the length of the sequence set forth in the identified
sequence, or by an articulated length (such as 100 consecutive
nucleotides or amino acid residues from a sequence set forth in an
identified sequence), followed by multiplying the resulting value
by 100. For example, a nucleic acid sequence that has 1166 matches
when aligned with a test sequence having 1154 nucleotides is 75.0
percent identical to the test sequence (1166/1554*100=75.0). The
percent sequence identity value is rounded to the nearest tenth.
For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to
75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to
75.2. The length value will always be an integer. In another
example, a target sequence containing a 20-nucleotide region that
aligns with 20 consecutive nucleotides from an identified sequence
as follows contains a region that shares 75 percent sequence
identity to that identified sequence (that is, 15/20*100=75).
[0069] For comparisons of amino 5 acid sequences of greater than
about 30 amino acids, the Blast 2 sequences function is employed
using the default BLOSUM62 matrix set to default parameters, (gap
existence cost of 11, and a per residue gap cost 5 of 1). Homologs
are typically characterized by possession of at least 70% sequence
identity counted over the full-length alignment with an amino acid
sequence using the NCBI Basic BLAST 2.0, gapped blastp with
databases such as the nr or swissprot database. Queries searched
with the blastn program are filtered with DUST (Hancock and
Armstrong, Comput, Appl. Biosci., 10:67-70, 1994). Other programs
use SEG. In addition, a manual alignment can be performed. Proteins
with even greater similarity will show increasing percentage
identities when assessed by this method, such as at least about
75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to a
protein.
[0070] When aligning short peptides (fewer than around 30 amino
acids), the alignment is performed using the BLAST2 sequences
function, employing the PAM30 matrix set to default parameters
(open gap 9, extension gap 1 penalties). Proteins with even greater
similarity to the reference sequence will show increasing
percentage identities when assessed by this method, such as at
least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence
identity to a protein. When less than the entire sequence is being
compared for sequence identity, homologs will typically possess at
least 75% sequence identity over short windows of 10-20 amino
acids, and can possess sequence identities of at least 85%, 90%,
95% or 98% depending on their identity to the reference sequence.
Methods for determining sequence identity over such short windows
are described at the NCBI web site.
[0071] One indication that two nucleic acid molecules are closely
related is that the two molecules hybridize to each other under
stringent conditions, as described above. Nucleic acid sequences
that do not show a high degree of identity may nevertheless encode
identical or similar (conserved) amino acid sequences, due to the
degeneracy of the genetic code, Changes in a nucleic acid sequence
can be made using this degeneracy to produce multiple nucleic acid
molecules that all encode substantially the same protein. An
alternative and not necessarily cumulative) indication that two
nucleic acid sequences are substantially identical is that the
polypeptide which the first nucleic acid encodes is immunologically
cross reactive with the polypeptide encoded by the second nucleic
acid.
[0072] One of skill in the art will appreciate that the particular
sequence identity ranges are provided for guidance only; it is
possible that strongly significant homologs could be obtained that
fall outside the ranges provided.
[0073] Subject:
[0074] A living multicellular vertebrate organism, a category that
includes, for example, mammals and birds. A "mammal" includes both
human and non-human mammals, such as mice. In some examples, a
subject is a patient, such as a patient that is HIV-positive
including a patient that is HIV-positive and undergoing
antiretroviral therapy (ART).
[0075] Test Compound:
[0076] A candidate molecule that is tested for its ability to
inhibit a CD4.sup.+ T cell latently infected with HIV, preferably
with minimal effects on normal CD4.sup.+ T cells. The test compound
can include any small organic molecule, or a biological entity. As
non-limiting examples, the test compound can be a protein (such as
an antibody or a peptide), a sugar, a nucleic acid (such as an
antisense oligonucleotide, a ribozyme, or RNAi molecule) or a
lipid. The test compound may be isolated, or may be part of a
mixture (for example two or more test compounds). The test compound
or mixture of test compounds may also include additional
components, such as diluents, solvents, pharmaceutically acceptable
carriers, or other compounds. Test compounds can also include
positive or negative controls known to inhibit or kill CD4.sup.+ T
cells.
[0077] Treating.
[0078] As used herein, the term "treating" refers its meaning as
known in the art, and to both therapeutic treatment and
prophylactic, or preventative, measures, or administering an agent
suspected of having therapeutic potential. The term includes
preventative (as a non-limiting example, prophylactic) and
palliative treatment. As used herein, the term "treatment" also
includes symptomatic therapy to lessen, alleviate, or mask the
symptoms of the disease or disorder, as well as therapy for
preventing, lowering, stopping, or reversing the progression of
severity of the condition or symptoms being treated. As such, the
term "treatment" includes both medical therapeutic treatment of an
established condition or symptoms and/or prophylactic
administration, as appropriate.
Methods of Identifying Cells with Latent HIV Nucleic Add
[0079] Disclosed herein are methods of identifying a cell from a
subject as having latent HIV nucleic acid. The methods involve
obtaining a biological sample from the subject. The biological
sample is contacted with one or more labeled antibodies. The
labeled antibodies are specific for PD-1, LAG-3, or TIGIT markers
and form a complex with said markers such that any cell in the
sample that expresses PD-1, LAG-3, or TIGIT will be bound by the
labeled antibody. The labels conjugated to said antibodies can be
detected by any appropriate method. Detection of the complex
signifies that the cell includes latent HIV nucleic acid. The use
of the combination of the labelled antibodies to PD-1, LAG-3, or
TIGIT markers to detect CDC cells with latent HIV nucleic acid is a
surprising effective diagnostic to distinguish patient prognosis
for HIV.
[0080] LAG-3 is also known as CD223, and can be detected with a
commercially available labelled antibody such as Human LAG-3
FITC-conjugated Antibody from polyclonal goat IgG (R&D Systems
#FAB2319F). Such a labelled antibody is antigen purified with a
specificity of less than 1% cross-reactivity with mouse LAG-3.
TIGIT is also known as Tg with ITIM domains, and is also known as
VSTM3 or WUCAM, and can be detected with a commercially available
labelled monoclonal antibody such as TIGIT-PerCP eFluor 710
(eBioscience #46-9500, clone MBSA43). PD-1 is also known as CD279
and can be detected with a commercially available monoclonal
labelled antibody such as Anti-human CD279 (PD-1) APC (eBioscience
417-9969-4).
[0081] The sample may be any sample that includes CD4.sup.+ T
cells, including blood, lymph, and/or a lymph node biopsy such as a
needle biopsy. Prior to contacting the biological sample with the
labeled antibody, the biological sample may be further processed,
for example by isolating mononuclear cells by centrifugation,
isolating CDC cells through cell sorting (fluorescent or magnetic)
or by any other processing method known in the art.
[0082] The labeled antibodies can be conjugated to a fluorescent,
chemiluminescent enzymatic, magnetic, metallic, chemical,
radioactive, or other label that signifies and/or locates the
presence of specifically bound antibody. In some aspects, the
conjugation can be achieved by the appropriate chemical linkage
(see Hermanson, G. T., Bioconjugate Techniques, 3.sup.rd Ed.,
Academic Press, 2013). In some aspects, the labeled antibodies can
be conjugated to a fluorescent compound such as fluorescein
isothiocyanate, rhodamine, phycoerythrin, phycocyanin,
allophycocyanin, a-phthaldehyde and fluorescamine or any other
appropriate fluorescent compound known in the art.
[0083] In some aspects of the invention, the label helps facilitate
isolating (interchangeably described as sorting or purifying) the
cells, for example through the use of fluorescent activated cell
sorting (FAN, sorting using magnetic beads, or through binding to
an affinity column.
[0084] The complex formation between a PD-1, LAG-3, or
TIGIT-binding agent and a PD-1, LAG-3, or TIGIT marker of the
present invention can be detected by measuring or visualizing
either the binding agent bound to the PD-1, LAG-3, or TIGIT marker
or unbound binding agent. Assays (immunoassays) of the invention
include competitive and noncompetitive ("sandwich") assays.
Immunoassays of the invention include but are not limited to assay
systems using techniques such as Western blots, radioimmunoassays,
ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays,
immunoprecipitation assays, precipitin reactions, gel diffusion
precipitin reactions, immunodiffusion assays, agglutination assays,
complement fixation assays, immunoradiometric assays, fluorescent
immunoassays, protein A immunoassays, flow cytometry or tissue
immunohistochemistry. Immunohistochemistry can be performed using a
PD-1, LAG-3, or TIGIT-binding agent (as non-limiting examples: an
antibody, antigen binding fragment thereof, or aptamer). In some
aspects, the antibody can be synthesized with a label (such as a
purification or epitope tag), or can be detectably labeled by
conjugating a label or label-binding group. In some aspects, a
chelator can be attached to the antibody. The antibody can then be
contacted to a histological preparation, as a non-limiting example,
a fixed section of tissue that is on a microscope slide. After an
incubation for binding, the preparation can be washed to remove
unbound antibody. The preparation can then be analyzed using
microscopy to identify if the antibody is bound to the preparation.
The method can be used to evaluate a cell or tissue sample. The
antibody (or other polypeptide or peptide) can be unlabeled at the
time of binding. After binding and washing, the antibody can be
labelled in order to render it detectable.
[0085] The detection of labeled antibodies in biological samples
can also be used to monitor the efficacy of potential anti-HIV
agents during treatment. In some aspects, the levels of labeled
antibodies to PD-1LAG-3, or TIGIT can be determined before and
during treatment.
[0086] The efficacy of the treatment agent can be followed by
comparing the expression of PD-1, LAG-3, or TIGIT or any mixture
thereof, throughout the treatment. Agents exhibiting efficacy are
those which decrease the level of a PD-1, LAG-3, or TIGIT marker as
treatment with the agent progresses.
[0087] In some examples, the sample is contacted with a second
labeled antibody including a second labeled antibody that
specifically binds CD4. For CD4, the label on the second antibody
is distinguishable from the label on the first antibody so as to
differentiate CD4.sup.+ cells latently infected with HIV from
CD4.sup.+ cells that do not. In some aspects, additional antibodies
may be used that bind to another 1CB. As a non-limiting example, a
first antibody that binds PD-1 and a second antibody that binds
LAG-3 can be used; or a first antibody that binds PD-1 and a second
antibody that binds TIGIT can be used; or a first antibody that
binds LAG-3 and a second antibody that binds TIGIT can be used; or
a first antibody that binds PD-1, a second antibody that binds
LAG-3, and a third antibody that binds TIGIT can be used. Any of
these combinations may be used with an antibody that binds CD4. Any
of these combinations can be used with an antibody that binds
another ICB such as BTLA, 284, CTLA4, TIM-3, or CD160. Any of these
combinations can be used with any other antibody with any other
specificity.
[0088] In some aspects, the method involves purifying the cells,
preferably through a method facilitated by the label conjugated to
the antibody. Such purification methods can include fluorescence
activated cell sorting or magnetic sorting.
[0089] This invention relates to CD4.sup.+ T cells latently
infected with HIV which are isolated by the disclosed methods.
These CD4.sup.+ T cells may be used for any of a number of purposes
including methods of screening for inhibitors of CD4.sup.+ T cells
latently infected with HIV. Such screening methods involve
contacting CD4.sup.+ T cells latently infected with HIV with a test
compound and assessing the ability of the test compound to inhibit
the CD4.sup.+ T cells latently infected with HIV. In some aspects,
the test compound is also contacted with control CD4.sup.+ T cells
that lack latent HIV nucleic acid. In some embodiments, the control
CD4.sup.+ T cells are from a healthy donor. In some embodiments,
the test compound is an antibody specific for PD-1, LAG-3 or TIGIT
markers.
[0090] In one aspect the invention provides for a method for the
diagnosis of HIV in a subject comprising: obtaining a biological
sample from the subject, the biological sample comprising CD4.sup.+
T cells; contacting the sample with a first labeled antibody
comprising a first label, wherein the first labeled antibody
specifically binds PD-1, LAG-3, or TIGIT; forming a complex between
the first labeled antibody to PD-1, LAG-3, or TIGIT with PD-1,
LAG-3, or TIGIT; detecting the first label on the surface of a
CD4.sup.+ cell that has bound the labeled antibody; wherein
detection of the first label indicates that the cell is a latently
infected CD4.sup.+ T cell.
[0091] In some aspects, the present invention relates to diagnostic
and prognostic methods for diseases such as HIV based on detection
of PD-1, LAG-3 or TIGIT markers in a subject. The method may be
validated by the use of a biological sample from a subject with HIV
and from age and gender matched controls, without HIV. A biological
sample which may contain CDC cells latently infected with HIV, such
as urine, blood, serum or plasma, is obtained from a subject having
or suspected of having HIV or suspected of being predisposed to
developing HIV. A corresponding body fluid may be obtained from a
subject that does not have HIV as a control.
[0092] In some aspects, the method of detecting the first label can
be performed using flow cytometry. In some aspects, the method can
comprise contacting the sample with a second labeled antibody,
wherein the second labeled antibody binds CD4. In some aspects, the
method can comprise isolating the CD4.sup.+ T cells that have bound
the first antibody. In some aspects, the isolation of the CD4.sup.+
T cells can be achieved with fluorescently activated cell sorting
or magnetic sorting.
[0093] The present invention relates to a population of isolated
CD4.sup.+ T cells. In some aspects, said cells can be isolated
using fluorescently activated cell sorting or magnetic sorting. In
some aspects, said cells can be isolated from a subject undergoing
antiretroviral therapy.
[0094] The present invention relates to a method of selecting a
compound that inhibits a latently infected CD4.sup.+ T cell by
contacting the isolated CD4.sup.+ T cell with a test compound and
assessing whether or not the test compound inhibited said CD4.sup.+
T cell from expressing PD-1, LAG-3, or TIGIT markers. In some
aspects, the isolated CD4.sup.+ T cell can be isolated by
fluorescently activated cell sorting or magnetic sorting. In some
aspects, the method can involve contacting a control CD4.sup.+ T
cell with the test compound wherein the control CD4.sup.+ T cell is
known not to comprise latent HIV nucleic acid. In some aspects, the
control CD4.sup.+ T cell is a CD4.sup.+ T cell from an HIV-negative
subject. In some aspects, the test compound can comprise an
antibody specific for PD-1, LAG-3, or TIGIT.
[0095] The preceding merely illustrates the principles of the
invention. It will thus be appreciated that those skilled in the
art will be able to devise various arrangements which, although not
explicitly described or shown herein, embody the principles of the
invention and are included within its spirit and scope.
Furthermore, all examples and conditional language recited herein
are principally intended expressly to be only for pedagogical
purposes and to aid the reader in understanding the principles of
the invention and the concepts contributed by the inventors to
furthering the art, and are to be construed as being without
limitation to such specifically recited examples and
conditions.
[0096] Although the invention has been described in terms of
exemplary embodiments, it is not limited thereto. Rather, the
appended claims should be construed broadly, to include other
variants and embodiments of the invention, which may be made by
those skilled in the art without departing from the scope and range
of equivalents of the invention.
EXAMPLES
[0097] The following examples are illustrative of disclosed
methods. In light of this disclosure, those of skill in the art
will recognize that variations of these examples and other examples
of the disclosed method would be possible without undue
experimentation.
Example 1
Methods
[0098] Expression of PD-1, CTLA-4, LAG-3, TIGIT, TIM-3, BTLA, 2B4
and CD160 were measured by flow cytometry on peripheral blood
mononuclear cells (PBMCs) from 48 subjects on ART for >3 years
with HIV viral load <50 copies/ml and with a CD4 count >350
cells/.mu.L. The frequencies of CD4 T cells harboring integrated
HIV DNA, total HIV DNA, 2-LTR circles and cell associated unspliced
(CA-US) HIV RNA were determined by qPCR. Integrated HIV DNA was
also measured in sorted memory CD4 T cell subsets expressing some
of these ICBs. More specifically, the impact of PD-1 engagement on
HIV reactivation was assessed in CD4.sup.+ T cells isolated from
virally suppressed subjects using beads coated with anti-CD3,
anti-CD28, PD-L1 or the appropriate control.
[0099] Antibodies and other reagents used: CD3-Alexa 700 (Becton
Dickinson #557943, clone UCHTI), CD4-Qdot605 (Invitrogen/Life
Technologies/Thermo Fisher #Q10008, clone S3.5), CD8-PacificBlue
(Becton Dickinson #558207, clone RPA-T8), CD45RA-APC H7 (Becton
Dickinson #560674, clone Hl 00), CD27-BV650 (Biolegend #302827,
0323), CCR7-PE Cy7 (Becton Dickinson #557648, clone 3012),
CD14-V500 (Becton Dickinson #561391, clone M5E2), CD19-Amcyan
(Becton Dickinson #339190, clone SJ25CI), Live/Dead Viability Aqua
Dead Cell Stain (Invitrogen #L34957), POI (CD279)-Alexa647 (Becton
Dickinson #560838, clone EH12.1), LAG3 (CD223)-FITC (R&D
systems #FAB2319F) TIGIT-PerCP eFluor710 (eBioscience #46-9500,
clone MBSA43).
Example 2
PD-1, LAG-3 and TIGIT in CD4.sup.+ T cells
[0100] Absolute CD4.sup.+ T cell counts were negatively correlated
with the expression of PD-1, LAG-3 and TIGIT in CD4 T-cells
(r=-0.53 p<0.0001, r=-0.51 p=0.0002, r=-0.40 p=0.005,
respectively). Interestingly, the frequency of CD4 T cells
harboring integrated HIV DNA was positively correlated with the
expression of these markers (r=0.29 p=0.06, r=0.31 p=0.04 and
r=0.46 p=0.002, for PD-1, LAG-3 and TIGIT respectively). With the
exception of TIGIT with 2-LTR circles (r=0.39 p=0.009), no
statistically significant associations were found between these
markers and total HIV DNA, 2-LTR circles or CA-US HIV RNA. Memory
CD4 T cells expressing high levels of PD-1 or LAG-3 were enriched
for HIV integrated DNA when compared to their negative
counterparts. Engagement of PD-1 with its ligand PD-L1 inhibited
viral production induced by TCR stimulation in latently infected
cells (mean=94.8% of inhibition), suggesting a functional role for
this receptor in the maintenance of HIV latency.
[0101] PD-1, LAG-3 and TIGIT have been identified as markers
associated with incomplete CD4.sup.+ T cell restoration and HIV
persistence during ART. The data further demonstrate that PD-1 and
LAG-3 identify cells carrying integrated HIV DNA in virally
suppressed subjects.
Example 3
ICBs Show Differential Expression in CD4 T Cells from Virally
Suppressed HIV Infected Subjects
[0102] In FIG. 1, the levels of expression of eight selected ICBs
(PD-1, LAG-3, TIGIT, CTLA-4, BTLA, CD160, 284, TIM-3) were measured
by multiparametric flow cytometry on CD4.sup.+ T cells from 48 HIV
infected subjects on suppressive ART for at least 3 years.
Example 4
The Percentage of CD4.sup.+ T Cells Expressing PD-1, LAG-3, and
TIGIT are Associated with Incomplete CD4 Restoration During ART
[0103] FIGS. 2A-C shows the association between the frequency of
CD4 T cells expressing PD-1, LAG-3, and TIGIT and the CD4 count was
addressed using a Spearman's rank correlation. Absolute CD4 T cell
counts were negatively correlated with the expression of PD-1,
LAG-3 and TIGIT in CD4 T-cells (r=-0.53 p<0.0001, r=-0.51
p=0.0002, r=-0.40 p=0.005, respectively).
Example 5
Integrated, Total, 2-LTR HIV DNA and UnSpliced HIV RNA are Detected
in CD4 T Cells from Virally Suppressed HIV Infected Subjects
[0104] FIG. 3 shows the results of quantifications of HIV DNA forms
(total, integrated and 2-LTR circles) performed by real time nested
PCR on total CD4 T cells lysed in proteinase K, as described in
Chomont N et al., Nat Med, 15, 893-900, 2009, incorporated by
reference herein. Cell associated US HIV RNA was quantified on
extracted RNA from CD4 T cells and normalized to 185 RNA.
Integrated, total, 2-LTR HIV DNA and US HIV RNA were detected in
98%, 100%, 80% and 100% of the samples, respectively.
Example 6
The Frequencies of CD4 T Cells Expressing PD-1, LAG-3, TIGIT are
Associated with HIV Persistence During ART
[0105] FIGS. 4A-C show that the frequency of CD4 T cells harboring
integrated HIV DNA was positively correlated with the expression of
PD-1, LAG-3 and TIGIT (r=0.29 p=0.06, r=0.31 p=0.04 and r=0.46
p=0.002). Correlations were determined with Spearman test.
TABLE-US-00001 SEQUENCE LISTING SEQ. ID NO: 1 is an amino acid
sequence of human PD-1 1 mqipqapwpv vwavlqlgwr pgwfldspdr
pwnpptfspa llvvtegdna tftcsfsnts 61 esfvlnwyrm spsnqtdkla
afpedrsqpg qdcrfrvtql pngrdfhmsv vrarrndsgt 121 ylcgaislap
kaqikeslra elrvterrae vptahpspsp rpagqfqtlv vgvvggllgs 181
lvllvwvlav icsraargti garrtgqplk edpsavpvfs vdygeldfqw rektpeppvp
241 cvpeqteyat ivfpsgmgts sparrgsadg prsaqplrpe dghcswpl SEQ ID NO:
2 is an amino acid sequence of human LAG-3 1 mweaqflgll flgplwvapv
kplqpgaevp vvwagegapa qlpcsptipl qdlsllrrag 61 vtwqhqpdsg
ppaaapghpl apgphpaaps swgprprryt vlsvgpgglr sgrlpiqprv 121
qldergrqrg dfslwarpar radageyraa vhlrdralsc rlrlrlgqas mtasppgslr
181 asdwvilncs fsrpdrpasv hwfrnrgqgr vpvresphhh laesflflpq
vspmdsgpwg 241 ciltyrdgfn vsimynltvl glepptpltv yagagsrval
pcrlpagvgt rsfltakwtp 301 paggpdllvt gdngdftlrl edvsqaqagt
ytchihlqeq qlnatvtlai itvtpksfgs 361 pgslgkllce vtpvsggerf
vwssldtpsq rsfsgpwlea qeaqllsqpw qcglyqgerl 421 lgaavyftel
sspgaqrsgr apgalpaghl llfltlgvls llllvtgafg fhlwrrqwrp 481
rrfsaleqgi hppqaqskie eleqepepep epepepepep epeql SEQ ID NO: 3 is
an amino acid sequence of human TIGIT 1 mrwcllliwa qglrqaplas
gmmtgtiett gnisaekggs iilqchlsst taqvtqvnwe 61 qqdqilaicn
adlgwhisps fkdrvapgpg lgltlgsltv ndtgeyfciy htypdgtytg 121
riflevless vaehgarfgi pllgamaatl vvictavivv valtrkkkal rihsvegdlr
181 rksagqeews psapsppgsc vqaeaapagl cgeqrgedca elhdyfnvls
yrslgncsff 241 tetg SEQ ID NO: 4 is an amino acid sequence of human
CD4 1 mnrgvpfrhl llvlqlallp aatqgkkvvl gkkgdtvelt ctasqkksiq
fhwknsnqik 61 ilgnqgsflt kgpsklndra dsrrslwdqg nfpliiknlk
iedsdtyice vedgkeevql 121 lvfgltansd thllqgqslt ltlesppgss
psvqcrsprg kniqggktls vsqlelqdsg 181 twtctvlqnq kkvefkidiv
vlafqkassi vykkegeqve fsfplaftve kltgsgelww 241 qaerasssks
witfdlknke vsvkrvtqdp klqmgkklpl hltlpqalpq yagsghltla 301
leaktgklhq evnlvvmrat qlqknltcev wgptspklml slklenkeak vskrekavwv
361 lnpeagmwqc llsdsgqvll esnikvlptw stpvqpmali vlggvaglll
figlgiffcv 421 rcrhrrrqae rmsgikrlls ekktcqcphr fqktcspi
Sequence CWU 1
1
41288PRTHomo sapiens 1Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val
Trp Ala Val Leu Gln 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu
Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro
Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr
Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp
Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90
95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile
Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala
Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala
His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln
Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu
Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala
Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu
Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215
220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro
225 230 235 240 Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe
Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser
Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp
Gly His Cys Ser Trp Pro Leu 275 280 285 2525PRTHomo sapiens 2Met
Trp Glu Ala Gln Phe Leu Gly Leu Leu Phe Leu Gln Pro Leu Trp 1 5 10
15 Val Ala Pro Val Lys Pro Leu Gln Pro Gly Ala Glu Val Pro Val Val
20 25 30 Trp Ala Gln Glu Gly Ala Pro Ala Gln Leu Pro Cys Ser Pro
Thr Ile 35 40 45 Pro Leu Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly
Val Thr Trp Gln 50 55 60 His Gln Pro Asp Ser Gly Pro Pro Ala Ala
Ala Pro Gly His Pro Leu 65 70 75 80 Ala Pro Gly Pro His Pro Ala Ala
Pro Ser Ser Trp Gly Pro Arg Pro 85 90 95 Arg Arg Tyr Thr Val Leu
Ser Val Gly Pro Gly Gly Leu Arg Ser Gly 100 105 110 Arg Leu Pro Leu
Gln Pro Arg Val Gln Leu Asp Glu Arg Gly Arg Gln 115 120 125 Arg Gly
Asp Phe Ser Leu Trp Leu Arg Pro Ala Arg Arg Ala Asp Ala 130 135 140
Gly Glu Tyr Arg Ala Ala Val His Leu Arg Asp Arg Ala Leu Ser Cys 145
150 155 160 Arg Leu Arg Leu Arg Leu Gly Gln Ala Ser Met Thr Ala Ser
Pro Pro 165 170 175 Gly Ser Leu Arg Ala Ser Asp Trp Val Ile Leu Asn
Cys Ser Phe Ser 180 185 190 Arg Pro Asp Arg Pro Ala Ser Val His Trp
Phe Arg Asn Arg Gly Gln 195 200 205 Gly Arg Val Pro Val Arg Glu Ser
Pro His His His Leu Ala Glu Ser 210 215 220 Phe Leu Phe Leu Pro Gln
Val Ser Pro Met Asp Ser Gly Pro Trp Gly 225 230 235 240 Cys Ile Leu
Thr Tyr Arg Asp Gly Phe Asn Val Ser Ile Met Tyr Asn 245 250 255 Leu
Thr Val Leu Gly Leu Glu Pro Pro Thr Pro Leu Thr Val Tyr Ala 260 265
270 Gly Ala Gly Ser Arg Val Gly Leu Pro Cys Arg Leu Pro Ala Gly Val
275 280 285 Gly Thr Arg Ser Phe Leu Thr Ala Lys Trp Thr Pro Pro Gly
Gly Gly 290 295 300 Pro Asp Leu Leu Val Thr Gly Asp Asn Gly Asp Phe
Thr Leu Arg Leu 305 310 315 320 Glu Asp Val Ser Gln Ala Gln Ala Gly
Thr Tyr Thr Cys His Ile His 325 330 335 Leu Gln Glu Gln Gln Leu Asn
Ala Thr Val Thr Leu Ala Ile Ile Thr 340 345 350 Val Thr Pro Lys Ser
Phe Gly Ser Pro Gly Ser Leu Gly Lys Leu Leu 355 360 365 Cys Glu Val
Thr Pro Val Ser Gly Gln Glu Arg Phe Val Trp Ser Ser 370 375 380 Leu
Asp Thr Pro Ser Gln Arg Ser Phe Ser Gly Pro Trp Leu Glu Ala 385 390
395 400 Gln Glu Ala Gln Leu Leu Ser Gln Pro Trp Gln Cys Gln Leu Tyr
Gln 405 410 415 Gly Glu Arg Leu Leu Gly Ala Ala Val Tyr Phe Thr Glu
Leu Ser Ser 420 425 430 Pro Gly Ala Gln Arg Ser Gly Arg Ala Pro Gly
Ala Leu Pro Ala Gly 435 440 445 His Leu Leu Leu Phe Leu Thr Leu Gly
Val Leu Ser Leu Leu Leu Leu 450 455 460 Val Thr Gly Ala Phe Gly Phe
His Leu Trp Arg Arg Gln Trp Arg Pro 465 470 475 480 Arg Arg Phe Ser
Ala Leu Glu Gln Gly Ile His Pro Pro Gln Ala Gln 485 490 495 Ser Lys
Ile Glu Glu Leu Glu Gln Glu Pro Glu Pro Glu Pro Glu Pro 500 505 510
Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Gln Leu 515 520 525
3244PRTHomo sapiens 3Met Arg Trp Cys Leu Leu Leu Ile Trp Ala Gln
Gly Leu Arg Gln Ala 1 5 10 15 Pro Leu Ala Ser Gly Met Met Thr Gly
Thr Ile Glu Thr Thr Gly Asn 20 25 30 Ile Ser Ala Glu Lys Gly Gly
Ser Ile Ile Leu Gln Cys His Leu Ser 35 40 45 Ser Thr Thr Ala Gln
Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln 50 55 60 Leu Leu Ala
Ile Cys Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser 65 70 75 80 Phe
Lys Asp Arg Val Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln 85 90
95 Ser Leu Thr Val Asn Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr
100 105 110 Tyr Pro Asp Gly Thr Tyr Thr Gly Arg Ile Phe Leu Glu Val
Leu Glu 115 120 125 Ser Ser Val Ala Glu His Gly Ala Arg Phe Gln Ile
Pro Leu Leu Gly 130 135 140 Ala Met Ala Ala Thr Leu Val Val Ile Cys
Thr Ala Val Ile Val Val 145 150 155 160 Val Ala Leu Thr Arg Lys Lys
Lys Ala Leu Arg Ile His Ser Val Glu 165 170 175 Gly Asp Leu Arg Arg
Lys Ser Ala Gly Gln Glu Glu Trp Ser Pro Ser 180 185 190 Ala Pro Ser
Pro Pro Gly Ser Cys Val Gln Ala Glu Ala Ala Pro Ala 195 200 205 Gly
Leu Cys Gly Glu Gln Arg Gly Glu Asp Cys Ala Glu Leu His Asp 210 215
220 Tyr Phe Asn Val Leu Ser Tyr Arg Ser Leu Gly Asn Cys Ser Phe Phe
225 230 235 240 Thr Glu Thr Gly 4458PRTHomo sapiens 4Met Asn Arg
Gly Val Pro Phe Arg His Leu Leu Leu Val Leu Gln Leu 1 5 10 15 Ala
Leu Leu Pro Ala Ala Thr Gln Gly Lys Lys Val Val Leu Gly Lys 20 25
30 Lys Gly Asp Thr Val Glu Leu Thr Cys Thr Ala Ser Gln Lys Lys Ser
35 40 45 Ile Gln Phe His Trp Lys Asn Ser Asn Gln Ile Lys Ile Leu
Gly Asn 50 55 60 Gln Gly Ser Phe Leu Thr Lys Gly Pro Ser Lys Leu
Asn Asp Arg Ala 65 70 75 80 Asp Ser Arg Arg Ser Leu Trp Asp Gln Gly
Asn Phe Pro Leu Ile Ile 85 90 95 Lys Asn Leu Lys Ile Glu Asp Ser
Asp Thr Tyr Ile Cys Glu Val Glu 100 105 110 Asp Gln Lys Glu Glu Val
Gln Leu Leu Val Phe Gly Leu Thr Ala Asn 115 120 125 Ser Asp Thr His
Leu Leu Gln Gly Gln Ser Leu Thr Leu Thr Leu Glu 130 135 140 Ser Pro
Pro Gly Ser Ser Pro Ser Val Gln Cys Arg Ser Pro Arg Gly 145 150 155
160 Lys Asn Ile Gln Gly Gly Lys Thr Leu Ser Val Ser Gln Leu Glu Leu
165 170 175 Gln Asp Ser Gly Thr Trp Thr Cys Thr Val Leu Gln Asn Gln
Lys Lys 180 185 190 Val Glu Phe Lys Ile Asp Ile Val Val Leu Ala Phe
Gln Lys Ala Ser 195 200 205 Ser Ile Val Tyr Lys Lys Glu Gly Glu Gln
Val Glu Phe Ser Phe Pro 210 215 220 Leu Ala Phe Thr Val Glu Lys Leu
Thr Gly Ser Gly Glu Leu Trp Trp 225 230 235 240 Gln Ala Glu Arg Ala
Ser Ser Ser Lys Ser Trp Ile Thr Phe Asp Leu 245 250 255 Lys Asn Lys
Glu Val Ser Val Lys Arg Val Thr Gln Asp Pro Lys Leu 260 265 270 Gln
Met Gly Lys Lys Leu Pro Leu His Leu Thr Leu Pro Gln Ala Leu 275 280
285 Pro Gln Tyr Ala Gly Ser Gly Asn Leu Thr Leu Ala Leu Glu Ala Lys
290 295 300 Thr Gly Lys Leu His Gln Glu Val Asn Leu Val Val Met Arg
Ala Thr 305 310 315 320 Gln Leu Gln Lys Asn Leu Thr Cys Glu Val Trp
Gly Pro Thr Ser Pro 325 330 335 Lys Leu Met Leu Ser Leu Lys Leu Glu
Asn Lys Glu Ala Lys Val Ser 340 345 350 Lys Arg Glu Lys Ala Val Trp
Val Leu Asn Pro Glu Ala Gly Met Trp 355 360 365 Gln Cys Leu Leu Ser
Asp Ser Gly Gln Val Leu Leu Glu Ser Asn Ile 370 375 380 Lys Val Leu
Pro Thr Trp Ser Thr Pro Val Gln Pro Met Ala Leu Ile 385 390 395 400
Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile 405
410 415 Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln Ala Glu Arg
Met 420 425 430 Ser Gln Ile Lys Arg Leu Leu Ser Glu Lys Lys Thr Cys
Gln Cys Pro 435 440 445 His Arg Phe Gln Lys Thr Cys Ser Pro Ile 450
455
* * * * *